GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis, is assigned to glycoside hydrolase family 4. We have previously hypothesized that this unusual enzyme catalyzes the hydrolysis of maltose 6-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In the past year our studies have provided physico-chemical, and kinetic evidence to support the postulated hypothesis. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium atom, consistent with C-H bond cleavage at each center being partially rate -limiting. Kinetic parameters were also determined for 1-2H-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Bronsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase that is also included in family 4 of the glycosyl hydrolase superfamily (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured, and the observation of very similar kcat values for all substrates including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This catalytic mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site. Remarkably, the sugar-6-phosphate product (glucose-6P), has the same anomeric (alpha) form in the two cases. A summary of our findings has recently appeared in the peer-reviewed journal, Biochemistry.